Engineered aryl sulfate-dependent enzymes

Abstract

The present invention provides several non-naturally occurring sulfotransferase enzymes that have been engineered to react with aryl sulfate compounds as sulfo group donors, instead of the natural substrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and with heparosan-based polysaccharides, particularly heparan sulfate, as sulfo group acceptors. Each of the engineered sulfotransferase enzymes have a biological activity characterized by the position within the heparosan-based polysaccharide that receives the sulfo group, including glucosaminyl N-sulfotransferase activity, hexuronyl 2-O sulfotransferase activity, glucosaminyl 6-O sulfotransferase activity, or glucosaminyl 3-O sulfotransferase activity. Methods of using the engineered sulfotransferases to produce sulfated heparosan-based polysaccharides, including polysaccharides having anticoagulant activity, are also provided.

We claim: 
     
         1 . A non-natural glucosaminyl 6-O sulfotransferase enzyme (6OST) enzyme engineered to have sulfotransferase activity in the absence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS), the sulfotransferase activity comprising the transfer of a sulfo group from an aryl sulfate compound to heparan sulfate to form a 6-O-sulfated heparan sulfate product. 
     
     
         2 . The non-natural 6OST enzyme of  claim 1 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises at least one amino acid sequence motif selected from the group consisting of SEQ ID NO: 257, SEQ ID NO: 260, SEQ ID NO: 291, and SEQ ID NO: 292. 
     
     
         3 . The non-natural 6OST enzyme of  claim 2 , wherein the non-natural 6OST enzyme has an amino acid sequence comprising multiple mutations relative to conserved amino acid residues found in natural 6OST enzymes within enzyme class EC 2.8.2.-, wherein:
 natural 6OST enzymes have sulfotransferase activity with heparan sulfate and a sulfo group donor, the sulfo group donor consisting of PAPS, to form the 6-O-sulfated heparan sulfate product; and   the amino acid sequence of the non-natural 6OST enzyme has at least 80% sequence identity with the amino acid sequence of a natural 6OST enzyme, the natural 6OST enzyme amino acid sequence selected from the group consisting of SEQ ID NO: 191, SEQ ID NO: 199, and SEQ ID NO: 201.   
     
     
         4 . The non-natural 6OST enzyme of  claim 3 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 257. 
     
     
         5 . The non-natural 6OST enzyme of  claim 4 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 260. 
     
     
         6 . The non-natural 6OST enzyme of  claim 5 , wherein the aryl sulfate compound is selected from the group consisting of p-nitrophenyl sulfate and 4-nitrocatechol sulfate. 
     
     
         7 . The non-natural 6OST enzyme of  claim 2 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 257. 
     
     
         8 . The non-natural 6OST enzyme of  claim 2 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 260. 
     
     
         9 . The non-natural 6OST enzyme of  claim 2 , wherein the aryl sulfate compound is selected from the group consisting of p-nitrophenyl sulfate and 4-nitrocatechol sulfate. 
     
     
         10 . A protein-substrate complex, the protein-substrate complex comprising an aryl sulfate compound bound within the active site of a non-natural glucosaminyl 6-O sulfotransferase enzyme (6OST) enzyme engineered to have sulfotransferase activity in the absence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS), the sulfotransferase activity comprising the transfer of a sulfo group from an aryl sulfate compound to heparan sulfate to form a 6-O-sulfated heparan sulfate product. 
     
     
         11 . The protein-substrate complex of  claim 10 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises at least one amino acid sequence motif selected from the group consisting of SEQ ID NO: 257, SEQ ID NO: 260, SEQ ID NO: 291, and SEQ ID NO: 292. 
     
     
         12 . The protein-substrate complex of  claim 11 , wherein the non-natural 6OST enzyme has an amino acid sequence comprising multiple mutations relative to conserved amino acid residues found in natural 6OST enzymes within enzyme class EC 2.8.2.-, wherein:
 natural 6OST enzymes have sulfotransferase activity with heparan sulfate and a sulfo group donor, the sulfo group donor consisting of PAPS, to form the 6-O-sulfated heparan sulfate product; and   the amino acid sequence of the non-natural 6OST enzyme has at least 80% sequence identity with the amino acid sequence of a natural 6OST enzyme, the natural 6OST enzyme amino acid sequence selected from the group consisting of SEQ ID NO: 191, SEQ ID NO: 199, and SEQ ID NO: 201.   
     
     
         13 . The protein-substrate complex of  claim 12 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 257. 
     
     
         14 . The protein-substrate complex of  claim 13 , wherein the aryl sulfate compound is selected from the group consisting of p-nitrophenyl sulfate and 4-nitrocatechol sulfate. 
     
     
         15 . The protein-substrate complex of  claim 11 , wherein the protein-substrate complex further comprises heparan sulfate, and the heparan sulfate is also bound within the active site of the non-natural 6OST enzyme. 
     
     
         16 . The protein-substrate complex of  claim 10 , wherein the protein-substrate complex further comprises heparan sulfate, and the heparan sulfate is also bound within the active site of the non-natural 6OST enzyme. 
     
     
         17 . The protein-substrate complex of  claim 16 , wherein the non-natural 6OST enzyme has an amino acid sequence comprising multiple mutations relative to conserved amino acid residues found in natural 6OST enzymes within enzyme class EC 2.8.2.-, wherein:
 natural 6OST enzymes have sulfotransferase activity with heparan sulfate and a sulfo group donor, the sulfo group donor consisting of PAPS, to form the 6-O-sulfated heparan sulfate product; and   the amino acid sequence of the non-natural 6OST enzyme has at least 80% sequence identity with the amino acid sequence of a natural 6OST enzyme, the natural 6OST enzyme amino acid sequence selected from the group consisting of SEQ ID NO: 191, SEQ ID NO: 199, and SEQ ID NO: 201.   
     
     
         18 . The protein-substrate complex of  claim 17 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 257. 
     
     
         19 . The protein-substrate complex of  claim 10 , wherein the aryl sulfate compound comprises a sulfuryl moiety, the sulfuryl moiety coordinated with a histidine residue engineered into the active site of the non-natural 6OST enzyme. 
     
     
         20 . The protein-substrate complex of  claim 19 , wherein the amino acid sequence of the non-natural 6OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 257, and the sulfuryl moiety is coordinated with the histidine residue of SEQ ID NO: 257.